Stroke is predicted to affect more than 600,000 people in the United States a year. In a 1999 report, over 167,000 people died from strokes, with a total mortality of 278,000. In 1998, 3.6 billion was paid to just those Medicare beneficiaries that were discharged from short-stay hospitals, not including the long term care for >1,000,000 people that reportedly have functional limitations or difficulty with activities of daily living resulting from stroke (Heart and Stroke Statistical update, American Heart Association, 2002). No therapeutics has yet been approved to reduce brain damage resulting from stroke.
Stroke is characterized by neuronal cell death in areas of ischemia, brain hemorrhage anti/or trauma. Cell death is triggered by glutamate over-excitation of neurons, leading to increased intracellular Ca2+ and increased nitric oxide due to an increase in nNOS (neuronal nitric oxide synthase) activity.
Glutamate is the main excitatory neurotransmitter in the central nervous system (CNS) and mediates neurotransmission across most excitatory synapses. Three classes of glutamate-gated ion channel receptors (N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and Kainate) transduce the postsynaptic signal. Of these, NMDA receptors (NMDAR) are responsible for a significant portion of the excitotoxicity of glutamate. NMDA receptors are complex having an NR1 subunit and one or more NR2 subunits (2A, 2B, 2C or 2D) (see, e.g., McDain, C. and Caner, M. (1994) Physiol. Rev. 74:723-760), and less commonly, an NR3 subunit (Chatterton et al. (2002) Nature 415:793-798). The NR1 subunits have been shown to bind glycine, whereas NR2 subunits bind glutamate. Both glycine and glutamate binding are required to open the ion channel and allow calcium entry into the cell. The four NR2 receptor subunits appear to determine the pharmacology and properties of NMDA receptors, with further contributions from alternative splicing of the NR1 subunit (Kornau et al. (1995) Science 269:1737-40). Whereas NR1 and NR2A subunits are ubiquitously expressed in the brain, NR2B expression is restricted to the forebrain, NR2C to the cerebellum, and NR2D is rare compared to the other types.
Because of the key role of NMDA receptors in the excitotoxicity response, they have been considered as targets for therapeutics. Compounds have been developed that target the ion channel (ketamine, phencyclidine, PCP, MK801, amantadine), the outer channel (magnesium), the glycine binding site on NR1 subunits, the glutamate binding site on NR2 subunits, and specific sites on NR2 subunits (Zinc—NR2A; Ifenprodil, Traxoprodil—NR2B). Among these, the non-selective antagonists of NMDA receptor have been the most neuroprotective agents in animal models of stroke. However, clinical trials with these drugs in stroke and traumatic brain injury have so far failed, generally as a result of severe side effects such as hallucination and even coma. Other criticisms of past animal stroke studies include that the efficacy of many neuroprotectants was determined in mild ischemia models (ischemia-reperfusion instead of permanent ischemia), and under conditions of food deprivation, which can not adequately mimic the more severe human situation. Also, most drugs were administered pre-ischemia whereas human trials necessitate a post-treatment paradigm (Gladstone et al., 2002; STAIR Committee, 1999).
Another key difference between human stroke and experimental ischemia is that some stroke victims also suffer from aggravating premorbid or comormid conditions or stroke-related complications. Prominent among these is hyperglycemia (Alvarez-Sabin, 2003), especially in diabetic patients (Paolino, 2005), but also in non-diabetics (Alvarez-Sabin, 2003). However, hyperglycemia is actively avoided in laboratory stroke studies as it is known to exacerbate cerebral infarction (Li, 1997, 1998, 2000, 2001; Farrokhnia, 2005), and experimental animals are routinely fasted to minimize intra-ischemic blood glucose elevations (Elsersy, 2004; Horiguchi, 2003; Belayev, 2005a). Fever is another complication that afflicts some stroke victims, and is an independent predictor of poor outcome (Azzimondi, 1995; Reith, 1996; Boysen, 2001; Ginsberg, 1998). Hyperthermia has long been known to exacerbate both global and focal experimental ischemic injury (Busto, 1987b, 1989a, 1989b; Ginsberg, 1992; Morikawa, 1992; Chen, 1993; Minamisawa, 1990a, 1990b, 1990c; Chen, 1991) and, precisely for this reason, has been strongly avoided in studies of neuroprotective drugs.
The present inventor has reported that postsynaptic density-95 protein (PSD-95) couples NMDARs to pathways mediating excitotoxicity and ischemic brain damage (Aarts et al., Science 298, 846-850 (2002)). This coupling was disrupted by transducing neurons with peptides that bind to modular domains on either side of the PSD-95/NMDAR interaction complex. This treatment attenuated downstream NMDAR signaling without blocking NMDAR activity, protected cultured cortical neurons from excitotoxic insults and reduced cerebral infarction volume in rats subjected to transient focal cerebral ischemia. The analysis was performed under conditions of transient ischemia and prior fasting to avoid exacerbating fever and hyperglycemia.